Mesenchymal Stem Cell-Derived Extracellular Vesicles in the Management of COVID19-Associated Lung Injury: A Review on Publications, Clinical Trials and Patent Landscape

The unprecedented COVID-19 pandemic situation forced the scientific community to explore all the possibilities from various fields, and so far we have seen a lot of surprises, eureka moments and disappointments. One of the approaches from the cellular therapists was exploiting the immunomodulatory and regenerative potential of mesenchymal stromal cells (MSCs), more so of MSC-derived extracellular vesicles (EVs)—particularly exosomes, in order to alleviate the cytokine storm and regenerate the damaged lung tissues. Unlike MSCs, the EVs are easier to store, deliver, and are previously shown to be as effective as MSCs, yet less immunogenic. These features attracted the attention of many and thus led to a tremendous increase in publications, clinical trials and patent applications. This review presents the current landscape of the field and highlights some interesting findings on MSC-derived EVs in the context of COVID-19, including in silico, in vitro, in vivo and case reports. The data strongly suggests the potential of MSC-derived EVs as a therapeutic regime for the management of acute lung injury and associated complications in COVID-19 and beyond. Graphical abstract

Introduction
Reporting more than 5.7 million deaths currently, COVID-19 (coronavirus disease 2019) a viral disease characterized by its high contagion caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a major impact on health systems worldwide (WHO Coronavirus (COVID-19) Dashboard [1]). COVID was first reported from a cluster of pneumonia of unknown origin in Wuhan, China in December 2019 [2]. A 13-fold increase in cases over the preceding timeline provoked WHO action and on March 11, 2020, WHO took the historic step of declaring this disease as a global pandemic [3]. Subsequently, the unbiased sequencing of pathogenic samples [4] from pneumonia-affected populations isolated a novel coronavirus signature [5]. The 2019-nCoV demonstrates a distinct difference from MERS CoV, SARS CoV and two other coronaviruses that infect humans. Common strains of Coronaviruses when infective to humans demonstrate common cold symptoms with no severe sequelae [6]. During the last 2003 SARS-CoV outbreak there were no traces of disease transmission before symptom onset [7]. Incisive detective work by several groups of researchers has determined that 40-80% of transmission of novel coronavirus disease happens before symptoms are demonstrated. This window may range from two to four days [8]. This has complicated public health measures aimed at the control of disease transmission necessitating debilitating lockdown measures in most parts of the world. In turn, it has precipitated the unprecedented spreading of the disease [9].
Therapeutic options that are currently available are antivirals, monoclonal antibodies, anti-inflammatory agents, immunomodulators with more on the way. Antiviral or antibody-based treatments are most effective during the early or replicative state of the disease. With later phases of the disease, it is ideal to seek control over inflammation, provide modulation as required and combine as required to achieve required endpoints [10]. A relatively new field, regenerative medicine has gained much popularity in the exploratory management of COVID-19. Regenerative medicine is accepted to be the culmination of multidisciplinary research and development that aims to be a definitive branch of medicine that will generate approaches to regenerate/repair defective tissues. A keystone of regenerative medicine is the application of mesenchymal stem cells (MSCs) derived from multifarious sources [11]. Adult stem cells are being commoditized for application in immunomodulation, angiogenesis, signaling and tissue remodeling applications [12]. The observation that during stem cell infusions effects are observed that are plausible only via secreted bioactive factors has been accepted [13,14]. Deployment to address Graft versus Host Disease [15] also reveals that most effects noted are due to paracrine cross-talk [16]. Long-distance communication has been via secretion of vesicles that range from 20 to 120 nm in size and function by transport of genetic material or protein signals [17]. The International Society of Extracellular Vesicles has proposed the generic term ''extracellular vesicles (EVs)'' for the membrane bound vesicles released from the cell, and typically, three primary classes of EVs are recognized based on size viz. apoptotic bodies ranging between -500 to 4000 nm, microvesicles ranging between -100 to 1000 nm, and exosomes ranging between -40 and 120 nm [18]. The use of EVs, particularly exosomes, has been explored in cardiovascular insults and cancer therapy. The use of these molecules as biomarkers and diagnostic tidemarks has been discussed and is under active investigation [19].
Exploratory studies on MSC-derived EVs, particularly exosomes, in the context of COVID-19 are growing recently. Briefly, MSC-derived EVs are being explored in various aspects of COVID19, including their use as biomarkers for diagnosis purpose, engineered EVs as immune-modulating agents, targeted therapeutic cargo as well as vaccines. The literature review indicated that various preclinical and clinical studies are underway with regard on finding an effective tissue source, effective dose, effective formulation, administrative dose, etc. Subsequent to completion of these studies, a comprehensive metaanalysis could derive an effective MSC-EVs based therapeutic regime for the management of acute lung injury in the context of COVID19. Several notable works have been reported recently on various aspects of stem cells and exosomes in the context of COVID19 [20][21][22][23]. However, this review standout as a unique collection of the current landscape in terms of preclinical, clinical and patent data sets reported to date.

COVID-19 and acute lung Injury
Based on the infection route and tissues affected, the COVID-19 disease progression can be represented as three phases [24]. Inhalation of virus particles causes the binding via Angiotensin-converting enzyme 2 (ACE2) receptor to the respiratory epithelial cells. ACE2 for entry and transmembrane serine protease 2 (TMPRSS2) for S protein priming have been pinpointed as its access and could be inhibited for clinical action [25]. Local propagation to the primary infection site and preliminary immune response initiation may happen in the initial days, with folks being asymptomatic carriers. Viral propagation and transition to lower respiratory tract along conducting airways and a more robust response from the host is generated over the next few days with clinical manifestation of the disease. Increased expression of Interferon-Inducible Protein-10 (CXCL-10) during the first 7 days of SARS was associated with an adverse outcome [26]. Assessing host immune response via monitoring of chemokine agents provides early insight to disease progression with [27] 80% of infected patients will have mild to moderate symptoms and can be managed with conservative therapy. In stage 3 of the disease affected parties may develop pulmonary infiltrates with a final fatality rate of around 2% with an agerelated distribution [24]. Stage 3 has disruption of gas exchange units and moves into alveolar type II cells which are typical in SARS medicated infections [28]. Diffuse alveolar damage with a disordered wound healing process is a precursor to scarring with attendant fibrosis [29] with any process that increases the ACE2 receptor population causing overall deterioration. With lessened secretory clearance and poor repair gas exchange units in the elderly are especially prone to non-reversible damage [30].
The acute lung injury that is the most severe aftershock of COVID-19 has no specific definitive treatment. Characteristics include lymphopenia and cytokine storm as part of immune pathological changes [31]. Host response via multiple streams contributes to damage in pulmonary tissue, reduction in total function and reduced lung capacity depending on lack of control over immune response [32]. T-cells on priming with SARS CoV-2 antigens kick off generation of chemokines and cytokines (IL-16,8,21, TNF-alpha) among others that sparks the cytokine storm. Recruitment of immune cellular component to the site of infection proceeds with nuclear factor-kappa B presence and activation being a strong predictor of immune response and inflammation associated respiratory tissue damage [33]. Subsets of severe COVID 19 diagnosed patients may present with elevated markers for inflammation, systemic inflammation and cascading multiple organ failure [33,34]. Cytokine storm in this disease context would refer to the dysregulation in cytokine release on systems being challenged with infection or other insults [34]. Increased IL-6 in plasma of acute cases of COVID 19 has been documented by several studies [35][36][37][38]. Elevated cytokine storm events contribute to pathological intra-alveolar fibrin accumulation and hikes production of growth factors [39] TGF, PDGF with a corresponding drop in surfactants in pulmonary tissue that causes coalescing alveoli leading to a decrease in effective lung volume [40]. This provides a niche conducive to fibroblast activity with rapid collagen deposition and the development of lung fibrosis [41]. A schematic showing pathophysiology of COVID-19 is presented in Fig. 1.

Stem Cells in COVID-19 and acute lung injury
In chronic and acute lung fibrosis there have been no treatment breakthroughs but with the current pandemic and its reach, there is a large fibrosis population that seeks to be addressed via bioengineering approaches. Damage in elderly or vulnerable populations [42] coupled with progenitor cell aging, accelerated senescence and aberrant regulation throws up multiple challenges in deciding therapeutic approaches [43]. Transient day-to-day insults to lung tissue are addressed by a resident progenitor cell population [44]. With aging, this gets more complicated with a complex interplay of elements with pathological levels of ECM deposition and enhanced circulating fibrocyte accumulation [45] in a bleomycin-induced lung injury model. Lowered reparative capacity hampers lung reserves and is dependent on the age and patency of cytokine response pathways [46]. Acute Respiratory Distress Syndrome (ARDS) based lung insults resulting in fibrosis are of concern under the current pandemic conditions. Incidence of ARDS although underreported was at 10-86 cases per 10,000 with a high fatality rate [47]. Currently, around 70% of mortality in COVID 19 has been directly linked to ARDS [48] with diffuse alveolar damage a hallmark in a majority of the cases [49].
Stem cells have been an accepted tool in the regenerative medicine approach due to their adaptability, ability to derive multiple cell types, compatibility with scaffold systems and also provision of extracellular vesicle-based therapies as the field progresses [50]. These undifferentiated cells survive within specialized niches in a self-renewing, clonogenic and multipotent state with the ability to activate and proliferate in response to stimuli within major organ systems [51]. A schematic representation of MSCs in acute lung injury management is depicted in Fig. 2. Several authors have reported the application of MSC could reduce severity in lung injury in preclinical models with a reduction in E. coli endotoxin-induced lung injury reported in mice [52]. Later reports reinforce MSC therapy in mice and ex vivo lung models challenged with live bacteria [53,54] with enhanced bacterial clearance and improvement in sepsis outcomes also reported [55,56]. Further investigation of MSC's mediation of repair has revealed the role of angiopoietin-1 [57], antagonists of interleukin-1 receptor and keratinocyte growth factor in this process [56]. Mitochondrial transfer mediated by exosomal vesicles is also postulated in this scenario [58,59]. Delivery of reparative signals from culturederived MSC extracellular vesicles would address these concerns without cell delivery. This simplifies the process and highlights a novel route to address ARDS and its longterm effects on respiratory tissue.

Stem cell-derived extracellular vesicles in COVID-19 and acute lung injury
Lightning advances in understanding the underlying mechanisms of MSC-based cellular therapy have shown their anti-inflammatory effects, regeneration ability, immune escaping characteristics and ability in modulating various other extra/intra -cellular pathways. Many of such Microvesicles with the size between 150 and 1000 nm and 3. Apoptotic bodies with greater size than the microvesicles. Initially they were misjudged as cellular waste and later found to be a functional vehicle that carry cargo to communicate neighbouring cells and influence various cellular signalling. The invasion of the endosomal membrane and fusion with membrane of multi-vesicular body leads to exosome formation, whereas microvesicles and apoptotic bodies are formed by outward budding of plasma membrane from healthy and apoptotic cells, respectively. EVs enclose various biologically active molecules, including various growth factors, cytokines, chemokines, miRNA, etc., however, their contents may vary, depending on the type of parental cell from which they are secreted and their environment. Nevertheless, the cell adhesion molecules present on the surface of EVs help them to fuse with the plasma membrane of the recipient cell so as to release the cargo and consequently trigger downstream signaling cascades [61]. In contrast to MSCs alone, the MSC-derived EVs offer many advantages as cell-free therapeutics such as high stability, easy storage, low immunogenicity and yet replicate the therapeutic effect of the MSCs [62,63]. A detailed account of EV's biogenesis, composition and functions are beyond the scope of this review, and the readers are referred to the latest review articles for the same [64][65][66][67].
The initial reports indicating the therapeutic potential of EVs in cardioprotection were published in 2009 [68]. Subsequently, several studies published various molecular and biochemical evidence to unravel the EVs mechanism of action in controlling viral replication, reducing the inflammatory reaction and regenerating the damaged tissues (Fig. 3). For instance, Kesimer et al. reported that EVs from airway epithelial cell cultures showed a neutralizing effect on the human influenza virus, therefore suggested that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense [69]. Other studies demonstrated that the EVs play a key role in suppressing the cytokine storm and rejuvenating the antiviral defense system of the host [61]. The EVs were reported to reduce the levels of virus-induced lung lesions including apoptosis in lung cells, by the production of growth factors such as keratinocyte growth factor, by reducing the influx of cytokines, inflammatory cells, by increasing the alveolar fluid clearance [70][71][72]. The MSC-EVs have the potential to repair and regenerate tissue, wherein some reports showed that the EVs induced the repopulation of type II alveolar cells which serve as progenitor cells for lung epithelium [73]. These and several other studies reviewed elsewhere offered possible mechanism of therapeutic action of MSC-derived EVs in the lung damage in general and in the COVID-19 management in particular [74][75][76][77][78]. Lately, The International Society for Cellular and Gene Therapies and the International Society for Extracellular Vesicles released a statement that they recognize, however do not endorse yet, the potential of EVs as treatments for COVID-19, and therefore encouraged research and trials in this regard [79].

Publications landscape
In silico studies The in silico approaches, such as docking, is well-regarded and validated for predicting new drug-target associations. Several computational studies are reported on the virtual drug repurposing screening and prediction of potential drug-like occurring molecules against SARS-CoV-2; however, many of them explore natural or other molecules known in the literature [80][81][82]. MSC-derived EVs were known to contain miRNAs that are being investigated for their role in the progression,  [77,[116][117][118] diagnosis and treatment of various diseases [83]. Several in silico studies are being undertaken to investigate the miRNA that can potentially aid in COVID-19 management (Fig. 4) [89,90]. Further, they demonstrated that EVs bearing hACE2 explicitly block the SARS-CoV-2 entry and thereby inhibit viral replication. The findings indicated a potential antiviral strategy involving EVs with an upregulated viral receptor that could competitively block the virus entry (Fig. 5). Although not related to MSC-EVs, in another interesting study, Wang et al. identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) in serum EVs that can directly inhibit S protein expression and SARS-CoV-2 replication [91]. However, these miRNA content has drastically decreased in serum exosomes in the elderly and diabetic patients. This study indicated that the varying levels of EVs-miRNA could be attributed to varying levels of treatment outcomes. Interestingly, there are not many in vitro reports on the MSCderived EVs in the context of COVID-19,perhaps, this could be due to the requirement of high containment laboratories and the risks involved.

In vivo reports
It is known that the in vitro models are cost-effective, relatively quick and offer control over test parameters, but unfortunately, they fail to mimic the native tissue physiology. In the context of COVID-19, several developments on in vivo models are underway in order to understand the mechanisms of pathogenesis and to establish the safety and efficacy of potential therapeutic agents and vaccines [92][93][94]. In the context of EVs in COVID-19 management, Fu and Xiong demonstrated the engineering of EVs tagged with S-protein receptor-binding domain (RBD) of SARS-CoV-2 and their ability to suppress SARS-CoV-2 infection in Transgenic mice that express human ACE2 (hACE2 mouse) (Fig. 5). The RBD domain plays a critical role in the adhesion, fusion and cellular entry of the virus. The study demonstrated the ability of RBD-tagged EVs, encapsulated with siRNAs, to recognize and target ACE2 protein on the surface of lung cells and suppress the infection of pseudovirus in hACE2 mice. They also showed that the RBD-tagged EVs were able to specifically target other organs such as kidneys, heart, etc., and therefore, these engineered EVs can be explored for the delivery of antiviral agents to treat multiorgan failure in SARS-CoV-2 [95].There are no in vivo studies on MSC-EVs in the in vivo models infected with the SARS-CoV-2 virus or its pseudo variants. However, many studies are reported on the use of MSC-EVs in animal models with acute lung injury induced by other agents like LPS [96][97][98][99][100].

Case reports
Owing to the rapid spread of COVID- 19

Clinical trials landscape
The first phase I clinical trial using ex vivo expanded bone marrow MSCs as cellular pharmaceutical agents in patients with hematologic malignancies was performed in 1995, followed by their wide use in various applications [105]. The registered clinical trials with MSCs from 2010 to December 2021, based on the data obtained from clinicaltrils.gov, shows a steady increase in the number of trials and therefore the increasing belief in the therapeutic potential of MSCs. Overwhelming in vitro and in vivo studies revealed that the MSCs suppress the over-activated inflammatory response, release factors including cytokines and extracellular vesicles, which promote neighbouring cell survival and growth and possess differentiation potential. In terms of lung regeneration, mainly, the immunomodulatory function of MSCs shown to help in the recovery of lung function and reduce pulmonary fibrosis [106]. From the completed studies, two studies involve the inhalation of EVs-based formulations at the concentration of 0.5-2 9 10 10 (twice a day during 10 days-Phase I and now enrolling for Phase II; NCT04491240) and 2 9 10 8 (5 times at Day 1, Day 2, Day 3, Day 4, Day 5-Phase I; NCT04276987) respectively. Another study involved intravenous administration of bone marrow MSC-derived EVs (800 Billion and 1.2 Trillion-Phase II; NCT04493242). AVEM Healthcare proposed to use intravenous administration of MSC-derived EVs as escalating dose of 2 9 10 9 , 4 9 10 9 , 8 9 10 9 /mL (phase I) and 8 9 10 9 , 4 9 10 9 , 8 9 10 9 /mL (phase II) every other day for a period of 5 days (NCT04798716). Athens medical society (Greece) and Tel-Aviv Sourasky Medical Center (Israel) registered the use of EVs that were engineered to overexpress CD24 from human embryonic kidney T-REx TM -293 cells (once daily by inhalation for 5 days-Phase II; NCT04969172 and NCT04747574). TC Erciyes University (Turkey) uses the exosome derived from COVID-19 specific T-cells, where the T-cells are activated in vitro with COVID-19 viral peptide fragments in the presence of cytokines (2 9 10 8 exosome five-time daily for 5 days-Phase I; NCT04389385).
In addition to MSC-derived EVs alone, Organicell Regenerative Medicine (USA) developed Zofin (an acellular, minimally manipulated product, derived from human amniotic fluid)-shown to have growth factors, cytokines, chemokines and other extracellular vesicles, and is in a clinical trial for COVID-19 (2-5 9 10 11 particles/mL of Zofin is administered intravenously on day 0, day 4 and day 8-Phase I and II; NCT04384445). It is therefore evident that the MSCs, as well as the MSC-derived EVs, have once again attracted huge attention as an effective cell therapy owing to the COVID-19 situation. It is anticipated that the outcome of these trials would yield hope in regenerative medicine, both in viral pneumonia conditions and beyond.

Patent landscape
Since the COVID-19 pandemic, several innovative developments happened towards COVID-19 management, both from diagnostic as well as therapeutic perspectives. Innovations in MSC-derived EVs have also come into the limelight and several interesting patent applications were filed. These innovations range from exploring EVs as biomarkers for diagnostics purposes, to the use of engineered EVs to counter SARS-Cov2 replication and to the use of EVs in lung regeneration. For instance, a human bone marrow-derived MSC line stably expressing human ACE2 protein was made and the method of extracting exosome rich in ACE2 protein was developed (patent no. CN112430581A). Similarly, the exosome expressing human ACE2 protein on the surface was made by inducing the HEK293T cells with IFN a/b, which in turn induces the expression of the ACE2 protein. The exosome rich in ACE2 on the surface could specifically interact with the RBD of spike (S) protein. The competitive inhibition-mediated binding of the exosome to the SARS-CoV2 could minimize the viral entry to human cells (patent no. CN112646781A). Another patent suggests that the vesicle Exosome-mediated vaccine development was proposed by making exosomes that express engineered S protein of SARS-COV2 fused with Vesicular Stomatitis Virus Glycoprotein (VSVG). The injection of this nano-scale exosome is proposed to act like a vaccine and stimulate antibody production (Patent No. CN112439058A). The delivery of EVs or exosome comprising a fragment of a viral peptide or virus interacting protein could induce the host immune response that act against virus (Patent No. WO2021181399A1). In another report, a recombinant plasmid was constructed with Spike-lamp2b and transfected in mouse dendritic cells. The secreted exosomes by the transfected cells were reported to carry S-protein and induce antibody synthesis while injecting into humans (Patent No. CN112111513A). On the other hand, Nanoantibody for S-protein that tightly combined with the spurt protein on the exosome was developed, which can inhibit the interaction between the spike protein and ACE2 receptor (Patent No. CN112626030A). The cargos of EVs from placental MSCs shown to have biomolecules that have inherent anti-inflammatory effect and miRNAs that efficiently disintegrates the RNA genome of coronavirus (WO2021225214A1). The exosome expressing recombinant SARS-CoV2 S-protein fused with VSVG was encapsulated with SARS-CoV2 siRNA, which could specifically inhibit the virus replication (Patent No. CN112226413A). Similarly, the miRNA targeting SARS-COV2 S-protein has enriched in NK cell-derived exosome, which could inhibit the virus replication (Patent No. CN111494416B). The suitable methods for delivering stable EVs to the lungs such as intranasal spray or intranasal nebulizer have been developed (patent no. CN113384597A and WO2021237100A1). Besides traditional vaccines and the new mRNA vaccine, these innovative developments could bring new avenues for the treatment and prevention of SARS-COV2 infection, further, the beneficial effects of MSC-derived EVs could be multitude by incorporating other biomolecules and drugs.

Summary and future prospects
Extracellular vesicles are membrane-bound sub-micron scale structures released into extracellular space by the cells in all living systems. It was recently shown that the production of several factors via EVs orchestrates the main modes of action of MSCs following infusion, and MSCderived EVs have a number of advantages over live cells, including the ability to reduce undesired side effects such as infusional toxicities. Therefore, MSC-EVs have received significant attention in both academia and industry, as reflected by a flare-up in the number of publications, clinical trials and patent applications. The reports demonstrated that MSC-derived EVs can control the inflammatory response, repair the alveolar damage and avert pulmonary fibrosis. As discussed in this review, MSC-EVs, both pristine as well as engineered, are now being explored for their use as biomarkers, immune-modulating agents, therapeutics as well as vaccines, in the context of COVID-19. The data shows that the studies are ongoing to find out the effective formulation such as injectable, inhalable, etc. Studies are ongoing with EVs from MSCs derived from various tissue sources viz. bone marrow, umbilical cord, etc. to dig deep into tissue-source dependant differences if any. Some other studies are ongoing to investigate the effective dose of EVs. Also, various routes of EVs administration viz. intravenous, nasal, etc. are being investigated.
Though current understanding of therapeutic potential mainly comes from pristine MSCs cultured under routine culture conditions, a variety of other approaches are under investigation by various groups mainly to enhance the potency of MSC-EVs. These futuristic approaches include specialized culture conditions, cellular reprogramming, nanotechnology enabled modification and more [107][108][109]. For instance, the MSCs may be subjected to various biophysical cues such as electric pulsing, mechanical cues, 2D For instance, bacteria-derived molecules like lipopolysaccharides and the cytokines are included in the culture medium to induce EVs primed with anti-inflammatory ability. Approaches involving overexpression of certain transcription factors, signalling molecules and miRNAs.
Bioengineering of EVs to enhance their potency is also being considered in two ways, firstly, by altering their tropism towards target cells, and secondly, by loading the EVs with desired cargo [110]. Nanotechnology based approaches are also under experimentation for engineering of smart EVs for precision medicine [111]. As with many cellular therapies, scale-up and clinical grade manufacturing of EVs is a significant process engineering challenge, however, efforts are on to overcome these barriers towards offering clinically meaningful MSC and EVs based therapies [112,113]. It is anticipated that, once the ongoing studies are completed, a comprehensive meta-analysis of the results would enable us to derive an effective MSC-EVs based therapeutic regime in the management of COVID-19 and beyond. Further, additional developments in enhancing the potency of EVs and in scaling-up of EVs production would surely make the EVs-based cell-free regenerative therapy a reality in the days to come.


extfieldsofstudy: Medicine Mathematics Physics

text:
Lotka–Volterra models for extraterrestrial self-replicating probes

A sufficiently advanced extraterrestrial civilization can send out a swarm of self-replicating probes for space exploration. Given the fast-growing number of such a probe, even if there is only one extraterrestrial civilization sending out such probes in the Milky Way galaxy, we should still expect to see them. The fact that we do not consist part of the Fermi paradox. The suggestion that self-replicating probes will eventually mutate to consume their progenitors and therefore significantly reduce the number of total probes has been investigated and dismissed in the literature. In this work, we revisit this question with a more realistic Lotka–Volterra model and show that mutated probes would drive the progenitor probes into “extinction,” thereby replacing them to spread throughout the galaxy. Thus, the efficiency of mutated probes in reducing the total number of self-replicating probes is even less than previously thought. As part of the analysis, we also suggest that, somewhat counter-intuitively, in designing self-replicating probes, one should not program them to stop replicating when sufficient mutation causes the probes to fail to recognize the progenitor probes as “self.”

The oscillatory number of the predator (y, the upper curve in red) and prey (x, the lower curve in blue) populations in the "plain-vanilla" predator-prey model. Here we use α 1, β 0.5, γ 0.2, δ 0.1. The initial conditions are x(0) y(0) 3 predator and prey SRPs with the Lotka-Volterra model. The conclusion reached was that this scenario cannot sufficiently reduce the prey SRPs, and therefore, the Fermi paradox remains unsolved.
As we will explain in the next section, there are some shortcomings in the assumptions in [23], and the modeling of predator-prey system with Lotka-Volterra equations require some care in what kind of terms should be included. We then propose a more realistic equation to study the species interaction. Our model makes it clear why predator SRPs cannot resolve the Fermi paradox.

Lotka-Volterra models for replicators
Aside from the migration effect from one star system to another, the Lotka-Volterra model assumed in [23] is essentially the standard "plain vanilla" form 1 : where x x(t), y y(t) denote the number of preys and predators, respectively. The coefficients α, β, δ, γ are assumed to be positive. They govern the growth rate of preys, the reduction in the prey population due to predation, the increase in the predator population due to predation, and the natural death rate of the predator species, respectively. One may ask why the natural death rate of the prey species is not included. The reason is straightforward: We could add such a term −α x (where α > 0), but in a healthy population, the growth rate should be larger than the death rate anyway, so the overall effect is (α − α )x, where α − α > 0. So it is easier to absorb α and just deal with α. As the coefficients are unknown, we are only interested in the qualitative features. The units for time t is therefore left unspecified.
The behavior of this ODE system is well-known: The two species are locked into oscillatory cycles, an example is shown in Fig. 1. As the number of predators increase, the prey population dwindles, and as the preys are reduced, the predators run out of food and they themselves start to die off. The prey population then recovers and the cycle renews. In the associated phase diagram, the only fixed point is a center (Fig. 2). While this model is rather simplified and suffers from some problems (such as the "attofox" problem [26]-the number of the species can get very small, which in the real world could have led to extinction rather than a recovery later on). However, as a first approximation, the crude Lotka-Volterra model does give a sensible description.
However, precisely because the population number oscillates, it is difficult to conclude whether the predator-prey scenario sufficiently reduces the number of total probes. It could be that we are near the "valleys" of the population number, when both predator and prey are relatively low in numbers. This is why the analysis in [23] needs to be so thorough as to considering migration effect. Intuitively this amounts to taking the observer (us) into account: what is the probability that at the corner of the galaxy, we see very few SRPs, given the oscillatory populations? It is remarkable that the result turns out to be inadequate to explain Fermi paradox.
In this work, we shall reconsider the application of the "plain vanilla" Lotka-Volterra equation. This requires some thoughts on the design of SRPs and how mutation might affect them. Assuming that errors are accumulated as the probes self-replicate (just like biological cell division, there might be programs in place to correct these errors, but errors are unavoidable), the new generations of SRPs would be expected to eventually be different from the original population in one way or another. In order to prevent the probes from consuming each other, the extraterrestrial engineers should design SRPs to still recognize mutated probes as self. Hence, a Fig. 2 The phase diagram of the "plain-vanilla" predator-prey model with α 1, β 0.5, γ 0.2, δ 0.1. There exists a center surrounded by closed orbits probe can only become predatory when the self-recognition feature breaks down due to a new mutation and no longer recognizes other variants as self.
Just like in [23], we will assume that the prey probes do not consume the predators. This assumption is not necessary. A predator is defined to be the population that benefits from the inter-species interaction and similarly the prey is the one that suffers. Therefore, the progenitor probe can prey on the predator probes, with some coefficient +β x y added into the Lotka-Volterra equation for dx/dt, but the overall coefficient β − β remains negative by definition of being a prey. Thus it is more convenient to just redefine −β + β → β, or equivalently assuming β 0. With the above, the distinction between predator and prey, as in Eq. (), can be made. In [23], the author mentioned the possibility of an "omnivorous" predator species that also consumes each other, and conclude that this should not affect the conclusion. Indeed, omnivorous predators are also replicating while consuming, so this makes sense. Thus, we can assume without loss of generality that predator species would recognize themselves and not become omnivorous. On the other hand, to replicate is the main purpose of SRPs. It is unlikely that the mutation into predator would also mutate the probes in so specific a way to make the predator probes exclusively consuming the prey probes. (In fact, as we will see, extraterrestrial engineers should try their best to program SRPs to prevent exclusive predation behavior from arising.) In other words, the Lotka-Volterra equation should include also the resource term for the predators. Unlike, say, foxes and rabbits in a typical ecosystem, in which the predators only eat the preys and would die off without the latter, SRPs dynamics would be quite different due to both species have access to environmental resources. This crucial term, missing in [23], makes all the difference. A more realistic model should therefore be (we use Latin coefficients in place of Greek ones for the newly considered terms): where A, B > 0. Here A is the coefficient of the resource term for the predator population. We expect that the magnitude of α is comparable to A, but they may not be identical after the mutation. (The result below holds as long as A > γ .) We have also included the mutation effect via the coefficient B, where preys are converted into predators with some fixed rate 2 . The magnitude of B should be much smaller than the other terms. The condition A > γ is simply the statement that the natural death rate is smaller than the growth rate of the population.
To solve for the fixed points, we solve the equations This yields one trivial fixed point (0, 0), as well as one nontrivial fixed point: We note that the nontrivial fixed point is irrelevant because x * < 0. The Jacobian associated to the dynamical system is  The origin (0, 0) is the only fixed point, and since J | (0,0) is a lower triangular matrix, its eigenvalues are α − B > 0, and A − γ > 0. The trivial fixed point is therefore an unstable node that repels all nearby trajectories in the phase space (Fig. 3). The result is that the prey quickly becomes extinct, while the predator population eventually grows exponentially (or tend to a constant if a finite capacity is assumed, à la the logistic model), as shown in the top plot of Fig. 4. The predator growth rate is comparable to the progenitor probe in the scenario in which no mutation occurs; see the bottom comparison plots of Fig. 4. We remark that the conversion term Bx is not crucial, the major influence is due to the resource term. Even if we take B 0 in the model (which corresponds to the mutation being a one-off event), the repelling behavior persists. From our discussion above, it seems that we do not have to discuss the A 0 case (which is excluded with our assumption that A > γ > 0). However, this case will provide some insights into how to design SRPs in the Discussion section and thus is worth some investigations. For A 0, the trivial fixed point (0, 0) is a saddle node. More crucially, the nontrivial fixed point is now relevant as x * , y * > 0. Though the system does not quite reduce to the "plain vanilla" Lotka-Volterra form (due to the Bx term in the dy/dt equation), for small enough B, we still have oscillatory behaviors not too different from the "plain vanilla" Lotka-Volterra equation, which requires a careful analysis of [23]. This is not surprising since the term Bx can be seen as a perturbation away from Fig. 5 The phase diagram of the predator-prey model with a "defective" predator. Here we use α 1, β 1, γ 5, δ 1, B 0.5, A 0. The system has a nontrivial fixed point marked with the red dot at (x * , y * ) (2.5, 0.5). In this case the trajectories are stable spirals, but this is not necessarily the case in general; see Fig. 6 Fig. 6 The phase diagram of the predator-prey model with a "defective" predator. Here we use α 1, β 0.5, γ 5000, δ 0.1, B 0.01, A 0. The system has a nontrivial fixed point marked with a red dot at (x * , y * ) ≈ (47596.15, 1.98), which is a stable node. The trajectories flow toward an attractor curve that leads to the stable node the "plain vanilla" model. As B gets larger, the nontrivial fixed point becomes an attractor. To see this, we note that the eigenvalues of J | (x * ,y * ) (A 0) are where it can be shown that Each of the terms in f (α, β, δ, B) are negative, so f (α, β, δ, B) < 0. Consequently we see that indeed for γ small enough, λ 1,2 are complex, whereas for large enough γ , both λ 1,2 < 0 so we have an attractor of the stable node type. For intermediate values of γ , we would obtain a stable spiral attractor instead. An example is provided in Fig. 5 with α 1, β 1, γ 5, δ 1 Figs. 3 and 4, γ needs to be of order O(10 3 ) in order to exhibit an attractor behavior. An example with γ 5000 that gives rise to a stable node is given in Fig. 6. Note that since the system is nonlinear, eigenvalue analysis for linear stability need not quantitatively yield the correct result for when exactly the center transits into spirals.
For our purpose, we note that since γ governs the predator population "death rate," this means that the attractor case corresponds to the predator probes being "defective," i.e., easily mulfunctioned. This could happen as part of the programming. We will return to this issue in the Discussion. (The possibility that SRPs eventually mutate so much they cease to replicate was also discussed in [29].) Note that the case of A 0 but A < γ is similar. and again since mutation would eventually give rise to new predators. The growth rate of the SRPs is therefore still exponential. This makes it clear that the Fermi paradox cannot be resolved.
This analysis also suggests a somewhat counter-intuitive way to program SRPs if we ever could use it to explore the Galaxy. We might be tempted to think that we should design them to stop foraging as soon as their self-recognition program becomes sufficiently faulty. For example, "self" can be defined as a neighborhood in the parameter space, so that a small mutation is still acceptable. A "kill" code that renders the probe unable to forage or/and inactive can be designed to switch on when a newly constructed probe mutates outside the acceptable margin. This mimics a biological scenario: A cell which has an error in its DNA that cannot be repaired may undergo self-destruction (apoptosis), though this does not always work, which could lead to cancer. This parallels the runaways production of predator probes in our case. It is possible that after mutation the kill code is not as effective as to render the probe immediately inactive, but still the code could increase the "death rate" of the probe.
Such a design, however, can be risky. If consuming resources is coupled with self-recognition, the two behaviors are not independent, which conceivably increases the chance that a mutation might affect both. A biological analogy is the mutation of a virus-when a virus mutates into a new variant that is more effective at spreading, its virulence may increase (or decrease) if this is coupled with transmissibility (which can either happen via a pleiotropic side effect or through genetic linkage), which is of course one current concern for COVID-19 [30]. For our case, a coupling between self-recognition and foraging behavior might not only cause a predator behavior in the first place, but also conceivably makes the predator solely feeds on the progenitor probes (A 0). In other words, such a design may increase the chance that an almost plain-vanilla predator-prey model emerges, at which point the growth rate of the probe population ceases to be exponential, and instead locked into oscillatory cycles, or tend to an attractor (if the predator is prey-specific and defective in the sense that its "death rate" is high). The best strategy is not to program anything, so that foraging and self-recognition stay as independent systems. If predation behavior occurs by chance, it is less likely to be progenitor-specific. The predator probes would then out-compete the progenitors and grow exponentially -which is the wanted behavior for SRPs. The risk of constructing SRPs is of course well-known and should be taken into account very carefully by any ambitious civilization [31].
In any case, it does seem that based on our work and that of [23], mutated probes cannot explain the Fermi paradox. Instead, one is faced with the possibility that the lack of SRPs might indicate that highly advanced extraterrestrial civilizations do not exist [32]. Another possibility is that the probes are around, we just have not detected them [12,33]. In fact, can we recognize a sufficiently advanced SRP technology if we see it? Of course, if SRPs do exist, their "ecosystem" might be very complicated depending on how they are programmed. Perhaps they are almost biological in the sense that mutation can lead to natural selection and vastly improved probes of different species can evolve throughout the generations. One also has to take into account that there are more than one extraterrestrial civilizations that are launching SRPs, which would treat each other as resources to be consumed. We can even entertain the science fiction possibility that an extraterrestrial civilization would anticipate rival probes in advance and program their SRPs to be able to evade enemies and/or defend themselves or, more interestingly, establish contact and collaborate.
Lastly, let us also mention the possibility of "information panspermia" first introduced by Gurzadyan [34]. This is the idea that information regarding the genomic sequence of an organism can be compressed by certain algorithm to be spread through the galaxy, either by transmitting (with the hope that some advanced civilization can download it) or by utilizing the SRPs, either by transmitting through the SRP network once it is established, or directly programmed into the SRPs. In the latter scenario, we can even envision that the probes from a sufficiently advanced civilization can "seed" life across the galaxy when they detected a suitable environment (hence conceivably recreating even an ecosystem instead of just a single species), or even "terraforming" a targeted planet. Indeed, instead of fleets of spaceships, it is much more convenient to colonize the galaxy in this way. Though mutation to the SRPs would not affect its spread as is evident in the above analysis, it is possible that the mutation could also lead to certain corruptions of the genetic information. Depending on the degree of the corruption (and the efficiency of the error correction codes), this could either render the mutated probe unable to perform their panspermia function, or just cause the life seeded in this way to be genetic variations of the original species. If the latter scenario happens, it would be evolution in action on the galactic scale.
All these considerations, albeit some are highly speculative, imply that simple models based on Lotka-Volterra equations are unlikely to tell the whole story with regards to the Fermi paradox. Nevertheless, lacking any knowledge about the technology and the psychology of extraterrestrial species, we should always start from understanding the simplest models.


Anesthetic management of patients with carnitine deficiency or a defect of the fatty acid β-oxidation pathway

Abstract Carnitine is essential for the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. The carnitine shuttle transports long-chain fatty acylcarnitine to the mitochondrial matrix. Subsequently, long-chain fatty acyl CoA, which is split from long-chain fatty acylcarnitine by carnitine palmitoyltransferase II, undergoes fatty acid β-oxidation. Acetyl CoA is produced from long-chain fatty acyl CoA via fatty acid β-oxidation and aids in the synthesis of adenosine triphosphate via the tricarboxylic acid cycle and electron transport chain. In addition, in the fasting state, it leads to ketone body production in the liver and glucose production via gluconeogenesis. However, patients with compromised fatty acid β-oxidation, owing to carnitine deficiency as well as defects in carnitine transport and the fatty acid β-oxidation pathway, develop hypoglycemia, cardiomyopathy, arrhythmia, and hypotonia. These conditions are attributed to the accumulation of released fatty acids and acylcarnitine. This review aimed to shed light on the anesthetic management of patients with compromised fatty acid β-oxidation undergoing various surgeries by assessing relevant case reports associated with fatty acid β-oxidation disorder in PubMed. Pre-anesthetic and intraoperative evaluation should include monitoring of glucose and carnitine levels and specific cardiac tests, such as echocardiography. Considering that propofol is dissolved in 10% long-chain fatty acids, propofol infusion should be avoided because of increased long-chain fatty acid loading in patients with compromised fatty acid β-oxidation. Thus, anesthesia using opioids (remifentanil and fentanyl), midazolam, dexmedetomidine, etomidate, and non-depolarizing neuromuscular blocking agents would be appropriate in such patients.

Introduction
Carnitine (b-hydroxoy-g-N-trimethylaminobutyric acid) is an essential water-soluble nutrient required for transporting longchain fatty acids from the cytoplasm to the mitochondrial matrix. [1] Dietary intake accounts for 75% of the total body carnitine in non-vegetarians, and the remaining 25% is endogenously produced from lysine and methionine in the liver and kidneys. [1] Despite the difference in dietary carnitine intake HKY and SHO contributed equally to this study. The funders had no role in the study design; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
between vegetarians and non-vegetarians, free plasma carnitine levels are constantly maintained within the reference limits (20-50 mM/L) because of renal resorption and excretion associated with an excess amount of carnitine. [1] The skeletal muscle contains more than 95% of the total body carnitine, and the remaining carnitine is present in the liver, heart, and kidneys. [1] Carnitine contributes to lipid metabolism and subsequent energy production via fatty acid b-oxidation in the heart, liver, and skeletal muscle. [1] Fatty acid b-oxidation is a physiological response to the energy shortage state of tissues that is caused by fasting, vigorous exercise, and stressful conditions (febrile illness, infection, and surgery). [2] In addition, fatty acid b-oxidation is associated with synthesis of adenosine triphosphate via the downstream tricarboxylic acid cycle and electron transport chain and meets 80% energy demand of the heart, liver, and skeletal muscle. [2] Thus, fatty acid b-oxidation disorder, which occurs because of carnitine deficiency and defects in the carnitine shuttle and fatty acid b-oxidation pathway, results in the development of hypoglycemia, cardiomyopathy, hepatomegaly, and rhabdomyolysis. [2] These disorders cause Reye-like syndrome, arrhythmia, cardiomyopathy, and muscle weakness in the fasting state during later infancy and childhood. [3] Rhabdomyolysis and cardiomyopathy due to fatty acid b-oxidation disorder are common adolescent-or adult-onset symptoms. [3] The wide range of clinical presentations and severity depends on the type or the management of the disease. In particular, poor control of fatty acid b-oxidation disorder causes severe damage to patients with hypoglycemia-induced brain damage. [1,4] The stress response to surgery and disease causes a derangement of metabolic and physiological responses, ultimately inducing hypercatabolism and hypermetabolism. [5] The ratio of primary carnitine deficiency is 1:142,000 in newborn screening tests; however, carnitine deficiency reportedly produces ventricular arrhythmia and hypotension induced by subtoxic doses of bupivacaine, thereby suggesting that the deficiency increases the susceptibility to bupivacaine-induced cardiac toxicity. [1,6,7] In addition, increased cortisol, catecholamine, and glucagon secretion because of surgery-induced stress response, which contributes to improved survival after trauma and surgery, stimulates lipolysis and ketone body production through fatty acid b-oxidation. [8] Thus, by assessing relevant previous reports, [1,[5][6][7][8] this review aimed to shed light on the importance of anesthetic management in patients with fatty acid b-oxidation disorder (carnitine deficiency and defects in the fatty acid b-oxidation pathway) who are undergoing various surgeries.

Methods
We searched for relevant articles in PubMed (date: March 11, 2021 to August 15, 2021) using the following terms: "carnitine and anesthesia" (N = 118), "carnitine deficiency and anesthesia" (N = 35), "carnitine acylcarnitine and anesthesia" (N = 11), "carnitine palmitoyltransferase deficiency and anesthesia" (N = 12), and "acyl-CoA and anesthesia" (N = 45). A total of 221 articles were retrieved from the database (Fig. 1). We excluded overlapping (N = 71) and not accessible (N = 4, written in Japanese or Spanish, not accessible full text) articles (Fig. 1). The remaining 146 articles were screened, of which 41 nonclinical case reports were excluded (Fig. 1). We carefully reviewed the remaining articles (N = 105). Articles on non-fatty acid b-oxidation disorder (N = 61) and those unrelated to anesthesia (N = 13) were also excluded ( Fig. 1). Eventually, we analyzed a total of 31 case reports (31 patients), with 1 case involving the same patient undergoing 2 surgeries (Fig. 1). Institutional review Full-text articles assessed for eligibility (n = 105) Records excluded • Overlapped same articles (n = 71) • Not accessible (n=4) Figure 1. Flow chart for extracting relevant case reports from PubMed on anesthesia for patients with fatty acid b-oxidation disorder who underwent surgery. We used the following terms: "carnitine and anesthesia," "carnitine deficiency and anesthesia," "carnitine acylcarnitine and anesthesia," "carnitine palmitoyltransferase deficiency and anesthesia," and "acyl-CoA and anesthesia." Yu et al.  101: 7 Medicine board approval is not necessary because this is an evidence-based narrative review.
Intraoperative and postoperative adverse events, including sudden cardiac arrest, sudden sinus bradycardia, ventricular arrhythmia, severe hypotension, and coma in the postanesthetic care unit, occurred in 4 cases (12.5%; Table 1). Of these cases, 2 patients had never been diagnosed with fatty acid b-oxidation disorder before the event and received only basic intraoperative monitoring and end-tidal carbon dioxide concentration during anesthesia. Treatment of this adverse event included cardiopulmonary resuscitation, epinephrine, ephedrine, atropine, carnitine, 20% glucose, lipid emulsion, and mechanical ventilation ( Table 1). Of the 4 cases involving severe adverse events, 2 patients recovered, and the remaining 2 patients with primary or secondary carnitine deficiency died (Table 1). Lipid emulsion was used to treat intractable hemodynamic instability due to bupivacaine toxicity in secondary carnitine deficiency, leading to reduced plasma bupivacaine concentration (Table 1). [7] It may be caused by increased susceptibility to bupivacaine toxicity in secondary carnitine deficiency due to valproic acid (Table 1). [7] One case with primary carnitine deficiency produced rhabdomyolysis and cardiac arrest following general anesthesia involving nitrous oxide, oxygen, and enflurane (Table 1). [11] 3. Discussion Carnitine deficiency is divided into 3 types as follows: primary carnitine deficiency, secondary carnitine deficiency, and defects in carnitine transport. Primary carnitine deficiency is caused by genetic disorder (autosomal recessive) of the organic carnitine transporter novel type 2 in the plasma membrane (Fig. 2), which contributes to carnitine transport. [1] Secondary carnitine deficiency is associated with enhanced renal excretion and inadequate dietary intake because of malabsorption, malnutrition, and www.md-journal.com Table 1 Characteristics of patients with fatty acid b-oxidation disorder who underwent anesthesia for various surgeries.
DFAO Hx.    drug treatments (e.g., valproic acid) (Fig. 2). [1] In addition, very long and medium chain acyl CoA dehydrogenase deficiency is associated with defects in the fatty acid b-oxidation pathway, and may produce secondary carnitine deficiency via the accumulation of acylcarnitine, thus leading to the inhibition of renal carnitine absorption (Fig. 2). [38] The defect in carnitine transport is attributed to the deficiency or inhibition of carnitine palmitoyltransferase (CPT) (-I and -II) and carnitine acylcarnitine translocase (CACT), which contribute to the carnitine shuttle involved in long-chain fatty acid transport to the mitochondrial matrix. [39] Long chain fatty acids are transported to the cytoplasm through fatty acid transport proteins in the plasma membrane. Subsequently, long-chain fatty acyl CoA synthase produces long-chain fatty acyl CoA from long-chain fatty acids (Fig. 2). [39] Long-chain fatty acylcarnitine, which is produced from long-chain fatty acyl CoA and carnitine by CPT-I, is transported from the cytoplasm to the intermembrane space of mitochondria (Fig. 2). [39] Consequently, long-chain fatty acylcarnitine gets transported from the intermembrane space of mitochondria to the mitochondrial matrix by CACT (Fig. 2). [39] Long-chain fatty acylcarnitine is split into long-chain fatty acyl CoA and carnitine by CPT-II in the mitochondrial matrix (Fig. 2), followed by the return of carnitine to the cytoplasm by CACT (Fig. 2). [39] Long-chain fatty acyl CoA undergoes fatty acid b-oxidation to produce acetyl CoA, which subsequently produces nicotinamide adenine dinucleotide hydrogen and flavin adenine dinucleotide via the tricarboxylic acid cycle, thereby leading to adenosine triphosphate production via the electron transport chain (Fig. 2). [2] The end stage of fatty acid   Figure 2. The pathway involving carnitine shuttle and subsequent fatty acid b-oxidation. Carnitine is transported to the cytoplasm via the organic carnitine transporter novel type 2 (OCTN2) in the plasma membrane. [1] Long chain fatty acid (LCFA) is transported to the cytoplasm via the fatty acid transport protein (FATP). [39] LCFA is converted to long chain fatty acyl CoA by long chain fatty acyl CoA (LCFA-CoA) synthase. [39] Long chain fatty acylcarnitine is produced from long chain fatty acyl CoA and carnitine by carnitine palmitoyltransferase I (CPT-I) in the inner side of the outer mitochondria membrane. [39] Long chain fatty acylcarnitine is transported from the cytoplasm to the intermembrane mitochondrial space, followed by the transport of long chain fatty acylcarnitine to the mitochondria matrix by carnitine acylcarnitine translocase (CACT). [39] It gets split into long chain acyl CoA and carnitine by carnitine palmitoyltransferase II (CPT-II) in the mitochondria matrix. [39] Carnitine is returned to the cytoplasm from the mitochondria matrix by CACT. [39] Long chain acyl CoA undergoes fatty acid b-oxidation mediated by 4 enzymes, including acyl CoA dehydrogenase, enoyl CoA hydratase, hydroxyacyl CoA dehydrogenase, and keto acyl CoA thiolase. [2] The first step of fatty acid b-oxidation is mediated by very long, medium, and short chain acyl CoA dehydrogenase, which involves electron transferring flavoprotein (ETF) and electron transferring flavoprotein dehydrogenase (ETFD). [2] Acetyl CoA is one of the final products of fatty acid b-oxidation, which produces nicotinamide adenine dinucleotide hydrogen (NADH)/flavine adenine dinucleotide (FADH2) via the tricarboxylic acid (TCA) cycle, thus leading to adenosine triphosphate (ATP) production. [2] In addition, acetyl CoA stimulates ketone and glucose production (via the activation of pyruvate carboxylase) in liver during fasting, thereby suggesting glucose sparing and glucose production, respectively. [41] Ketone is produced by acetyl CoA during fasting in the liver and is used as alternative energy source in the skeletal muscle, heart, and brain during fasting, thus suggesting glucose sparing. [41] However, the inhibition of acetyl CoA production caused by compromised fatty acid b-oxidation, which occurs because of carnitine deficiency (primary, secondary: because of an inborn error of metabolism or valproic acid) or the defect of carnitine transport (CPT-I or CPT-II deficiency) or fatty acid b-oxidation pathway (because of various acyl CoA dehydrogenase deficiency or a deficiency of ETF and ETFD), leads to cardiac arrhythmia and cardiomyopathy (heart), hypotonia and rhabdomyolysis (muscle), steatosis (liver), hypoglycemia, and the loss of consciousness (brain). [1,2,39,41] Yu et al. Medicine (2022) 101:7 www.md-journal.com b-oxidation produces acetyl CoA from long chain fatty acyl CoA, which is mediated by 4 enzymes-namely, acyl CoA dehydrogenase, enoyl CoA hydratase, hydroxyacyl CoA dehydrogenase, and ketoacyl CoA thiolase (Fig. 2). [2,40] Thus, defects in the fatty acid b-oxidation pathway (N = 18), secondary carnitine deficiency (N = 5), primary carnitine deficiency (N = 4), and defects in carnitine transport (N = 4) inhibits fatty acid b-oxidation. [2] In addition, ketone body (b-hydroxybutyrate, acetoacetate, and acetone) production by acetyl CoA generated by fatty acid b-oxidation in the liver during fasting is a predominant substitute for energy production in the heart, skeletal muscle, and brain (Fig. 2). [41] The very long, medium, and short chain acyl CoA dehydrogenase (Fig. 2), which is dependent on the carbon number of fatty acids, is involved in the first step of fatty acid b-oxidation from long-chain fatty acyl CoA. [2] Electron transferring flavoprotein and electron transferring flavoprotein dehydrogenase (Fig. 2), which contribute to the first step of fatty acid b-oxidation by very long, medium, and short chain acyl CoA dehydrogenase, are associated with the electron transport chain. [2,40] Carnitine deficiency and defects in the downstream fatty acid b-oxidation pathway can affect the liver, heart, skeletal muscle, and brain by the accumulation of fat released from adipose tissues during fasting, which leads to steatosis (liver), cardiomyopathy (including arrhythmia), myopathy (including hypotonia and rhabdomyolysis), and the loss of consciousness (brain) because of hypoglycemia (Fig. 2). [41] Furthermore, reduced acetyl CoA production caused by an inhibition of fatty acid b-oxidation during fasting prevents the production of ketones, which can be used as an alternative energy source (Fig. 2). [41] This inhibition is an outcome of carnitine deficiency or defects in either carnitine transport or fatty acid b-oxidation pathway. In addition, acetyl CoA stimulates pyruvate carboxylase to activate gluconeogenesis in the liver, thus leading to the production of glucose (Fig. 2). [41] Taken together, acetyl CoA production via fatty acid b-oxidation during fasting contributes to both glucose production via gluconeogenesis and glucose sparing via ketone production as an alternative energy source (Fig. 2). [41] Thus, the inhibition of acetyl CoA production inhibits gluconeogenesis and subsequently produces hypoketotic hypoglycemia and the loss of consciousness in the brain (Fig. 2). [41] Furthermore, considering the use of long-chain fatty acids as an energy source (energy: 80%) in the heart, carnitine deficiency and subsequent fatty acid b-oxidation inhibition lead to cardiomyopathy and myocardial depression by the accumulation of the released fatty acid and acylcarnitine. [2,39,41] In addition, glutaric aciduria type II (N = 3; Table 1) because of the deficiency of electron transferring flavoprotein and electron transferring flavoprotein dehydrogenase produces fatty acid b-oxidation disorder. [40,42] Bupivacaine reportedly inhibits CACT, which contributes to carnitine shuttle associated with long-chain fatty acid transport to the mitochondrial matrix. [43] The pre-anesthetic laboratory examination in patients with carnitine deficiency or defects in the fatty acid b-oxidation pathway should include electrocardiogram, echocardiography, blood glucose, carnitine, creatine kinase, and serum transaminase ( Table 1). Patients with primary carnitine deficiency have low levels of carnitine in the plasma (<5 mM/L), which necessitates life-long L-carnitine (100-200 mg/kg/day) treatment, which leads to the prevention of hypoglycemia and the improvement of cardiomyopathy and muscle weakness. [1,39] However, considering the inefficacy of carnitine treatment in the defect of carnitine transport, carnitine treatment was not performed in such cases (N = 4; Table 1). Carnitine deficiency and defects in the fatty acid b-oxidation pathway necessitate hypoglycemia prevention by frequently eating and avoiding fasting. Therefore, glucose (5% and 10%) was pre-operatively used for the prevention of intraoperative hypoglycemia in 22 cases (68.75%; Table 1). [1] Considering that fatty acids are major energy sources in catabolic states, such as fasting, vigorous exercise, infection, and surgery, the long-term goal of glucose supply, which prevents hypoglycemia in the acute stage, is to inhibit further fatty acid b-oxidation in patients with compromised fatty acid b-oxidation. [41] This eventually results in the decreased release of fatty acids from adipose tissues. [41] The pre-operative evaluation should focus on the following factors in patients with carnitine deficiency or defects in the fatty acid b-oxidation pathway (Table 1): the optimization of glucose level worsened by pre-operative fasting and stress, an evaluation of cardiac function, including echocardiography, and L-carnitine treatment (in carnitine deficiency). The pre-operative diagnosis of most patients (N = 28; 90.3%; Table 1) with fatty acid b-oxidation disorder warrants recording medical history. In addition, clinicians should document the family history (N = 6, 19.3%) associated with fatty acid b-oxidation disorder and other medication history (e.g., valproic acid), which can cause secondary carnitine deficiency and defects in fatty acid b-oxidation ( Table 1).
Considering that propofol is dissolved in 10% intralipid containing 100% long-chain fatty acid, it should be avoided in patients with carnitine deficiency or defects in the fatty acid b-oxidation pathway. This is because propofol increases longchain fatty acid loading in patients with compromised fatty acid b-oxidation. [25,29] Thus, we surmised that only 6 (18.75%) of the 32 surgeries used propofol as an induction or maintenance agent of general anesthesia (Table 1). [7,12,13,20,28,30] Furthermore, the probability of the propofol infusion syndrome increased upon the intravenous administration of propofol (>4 mg/kg/h) for more than 48 hours. [44] The suggested underlying mechanism of propofol infusion syndrome includes the inhibition of CPT-I, fatty acid b-oxidation, and electron transport chain, similar to an acquired form of carnitine deficiency. [45,46] Thus, it would be feasible to avoid propofol in patients with compromised fatty acid b-oxidation, notwithstanding small dosage and short infusion duration. [45,46] However, total intravenous anesthesia using propofol was performed in 3 cases (9.37%; Table 1). [12,20,30] One case underwent a bolus administration of 2 mg/kg propofol, followed by 8 mg/kg/h propofol for 3 hours. [12] The remaining 2 cases involved continuous infusion (0.08 mg/kg/ min and 0.016 mg/kg/min), which were supposedly small doses. [20,30] In addition, total intravenous anesthesia or sedation, which used ketamine-fentanyl, remifentanil-dexmedetomidinemidazolam, or midazolam-alfentanil other than propofol, was performed in 4 patients with compromised fatty acid b-oxidation (Table 1). [24,31,33,34] Considering CPT deficiency in some patients susceptible to malignant hyperthermia, the use of depolarizing muscle relaxant and inhalational anesthetic agent (sevoflurane, desflurane, and isoflurane) should be avoided in such patients. [47,48] Dantrolene should be prepared to treat malignant hyperthermia. Thus, 3 patients of 4 patients with CPT-I or CPT-II received neuraxial block (Table 1). However, 1 patient with CPT-I deficiency underwent general anesthesia with a volatile anesthetic (sevoflurane) and depolarizing neuromuscular blocker (suxamethonium), and was admitted to the intensive care unit in coma status following adenoidectomy. [17] At admission, she did not inform about being diagnosed with CPT-I deficiency; Yu et al.  101: 7 Medicine however, she recovered completely from coma after being treated with carnitine, dextrose, and oxygen. [17] Regional anesthesia can be used to avoid malignant hyperthermia in patients with CPT deficiency. As the non-toxic dose of bupivacaine produces ventricular arrhythmia and hypotension in patients with secondary carnitine deficiency who exhibit plasma carnitine levels within or below the reference limits, a subtoxic dose of local anesthetic-induced cardiotoxicity should be considered cautiously. [6,7,14] In addition, glucose infusion and intraoperative glucose monitoring should be maintained under regional anesthesia. Among all surgeries performed in patients with fatty acid b-oxidation disorder, deliveries accounted for the largest proportion (N = 5; 15.6%). Regional anesthesia, such as epidural anesthesia for delivery that includes careful cardiac monitoring, is reasonable in patients with CPT deficiency. Compared with the muscle carnitine levels before treatment, the levels were increased by the 6 months supply of L-carnitine in patients with infantile lipid myopathic carnitine deficiency; however, some patients did not reach the normal carnitine level in muscles. [49] This necessitates an adequate amount of L-carnitine for a sufficient period to reach adequate levels in the skeletal muscle, heart, and liver. In addition, the plasma free carnitine level does not always reflect the tissue level. [6,50] Intraoperative monitoring, which includes blood glucose, electrolyte, acid-base balance, creatine kinase, and cardiac monitoring, such as intraoperative echocardiography, is essential for patients with carnitine deficiency or defects in the fatty acid b-oxidation pathway. In particular, glucose is intravenously administered during the perioperative period, including fasting time. Moreover, glucose levels should be monitored intraoperatively. Secondary carnitine deficiency due to a deficiency of 3-methylcrontonyl-CoA carboxylase or isovaleryl-CoA dehydrogenase (Table 1), which is involved in leucine metabolism, increases the excretion of carnitine via carnitine conjugation (e.g., 3-hydroxyisovalreylcarnitine) (Fig. 2). [6,13,51] Symptoms of secondary carnitine deficiency are mild, compared to primary carnitine deficiency. [1] The prevention and treatment of hypoglycemia is important in patients with defects in the fatty acid b-oxidation pathway, such as very long and medium chain acyl CoA dehydrogenase deficiency. However, L-carnitine supplementation can be attempted in very long and medium chain acyl CoA dehydrogenase deficiency after low plasma carnitine is confirmed. [52,53] Thus, carnitine supply observed in 6 patients with defects in the fatty acid b-oxidation pathway (N = 6; 19.3%; Table 1) may be associated with the treatment of secondary carnitine deficiency. Clinically relevant concentrations of inhalation anesthetics (halothane, isoflurane, and sevoflurane) slightly inhibit complex I of the electron transport chain. [54] In addition, enflurane increases the fatty acid plasma concentration. [55] Hence, the use of intravenous anesthesia with opioids, benzodiazepines, dexmedetomidine, etomidate, and non-depolarizing muscle relaxants would be reasonable in patients with compromised fatty acid b-oxidation.

Conclusions
In summary, frequent eating, short duration of fasting, and stress reduction (infection and fever) are essential to avoid hypoglycemia. Pre-operative evaluation should include plasma glucose and carnitine levels to confirm whether they are within the reference limits. Dextrose (5% and 10%) is intravenously administered during the pre-operative period, and glucose should be monitored pre-operatively, intraoperatively, and postoperatively. More-over, general anesthesia using inhalation anesthetics and succinylcholine should be avoided in patients with CPT deficiency to prevent the occurrence of malignant hyperthermia. A subtoxic dose of local anesthetic may cause cardiotoxicity because of increased susceptibility to local anesthetic systemic toxicity in patients with carnitine deficiency. This warrants meticulous monitoring, including cardiac monitoring, during regional anesthesia in patients with compromised fatty acid b-oxidation. Moreover, propofol infusion should be avoided because propofol may not only induce propofol infusion syndrome but also increase long-chain fatty acid loading in patients with compromised fatty acid b-oxidation. Intravenous anesthesia using opioids (fentanyl and remifentanil), benzodiazepine (midazolam), etomidate, and dexmedetomidine would be more suitable for patients with compromised fatty acid b-oxidation.